The long-term objective of this application is to gain a better understanding of the physiological roles(s) played by 5-HT2CRs in the CNS. 5-HT2CRs are widely distributed throughout the CNS. Several studies have provided evidence implicating 5-HT2CRs in appetite regulation. Agonist-induced activation of postsynaptic 5-HT2CRs causes hypophagia in starved mice, while transgenic mice lacking functional 5-HT2CRs are obese. 5-HT2CRs activate phosphatidylinositol (PI) hydrolysis by interacting with G proteins. Recently, somatic mutations or polymorphisms in GPCRs have been identified in several diseases, including cancer. These mutations render the receptors constitutively active (capable of activating intracellular events in the absence of agonist). These observations unite to form an intriguing hypothesis that altered 5-HT2CR function may play a role in eating disorders, leading to obesity in the case of inactivation or anorexia in the case of overactivation. In order for the hypothesis to be valid, first it must be demonstrated that 5-HT2CRs can be rendered constitutively active. Preliminary studies from our laboratory indicate that mutation of a single amino acid in the third intracellular loop of the 5-HT2CR confers constitutive activity. Therefore, the specific aims of this application involve testing the hypothesis that 5-HT2CRs can be rendered constitutively active by specific amino acid substitutions within critical domains of the receptor. This will be accomplished using site-directed mutagenesis to create mutant receptors. Constitutive activity will be monitored using radioligand binding and PI hydrolysis to demonstrate increased agonist affinity, potency, and second messenger activation in the absence of agonists (hallmark features of constitutively active GPCR). Constitutively active receptors will be expressed in mammalian cells to determine if the constitutive activity is relevant at the cellular level. Antagonists will be tested for inverse agonist activity (ability to reverse constitutive activity of mutant 5-HT2CRs). These studies will enhance our understanding of 5-HT2CR function at the molecular level, provide a new model system for exploring the role of 5-HT2CRs in appetite regulation, and may lead to the development of a novel class of therapeutics for regulating appetite.